Abstract Epigenetic modifications to the genome, including methylation (mC) and hydroxymethylation (hmC) of cytosine bases regulate genome availability and gene expression. In the CNS, proper regulation of DNA modifications is critical to brain structure, function, and memory formation. Alterations to genomic patterns of mC/hmC are evident with aging and are hypothesized to cause age-related cellular dysfunction. However, the impact of age-related altered mC/hmC genomic patterns in the CNS on cognitive decline and neurodegenerations such as Alzheimer?s is unknown. At the tissue level, we have identified that the changes in mC/hmC patterns with age are largely sexually divergent and predominantly occur in non-canonical CH sites (where H is any base but G) in the hippocampus, unlike other, mitotic, tissues. We have also shown that age-related changes in mC can be prevented by life-long caloric restriction. However, it is well established that the CNS gene expression response to aging is cell-type specific and no studies of DNA modifications in isolated cell types with aging have been performed. Therefore, it is critical to study changes in mC and hmC with aging across the whole genome in a cell-type specific manner. Also, given the molecular and clinical sex divergences with aging, these studies must examine both sexes. Our long-term goal is to prevent or reverse age-related changes in DNA modification patterns to maintain brain function and prevent neurodegenerative disease. The objective of this work is to identify the cell- and sex-specific alterations to DNA modification patterns with brain aging and the regulatory mechanisms causing changes at these specific genomic loci. Our rationale is that the field does not currently know how these patterns change with aging and until these patterns are understood, interventions, such as epigenome editing or modulation of targeting mechanisms, cannot be developed. Our specific aims will: 1) evaluate hippocampal mC/hmC patterns in microglia, astrocytes and neurons across the genome with aging in male and female mice, 2) test whether a ?youthful? modification pattern can be rejuvenated by heterochronic parabiosis in old mice, and 3) examine how age-related modification patterns are targeted to specific genomic locations and identify how altered DNA modification patterns regulate gene expression. The significance of this work is that it will enable the field to move from correlative analyses to mechanistic studies that unravel the function of epigenetic changes with brain aging and the molecular processes that drive them. The proposed research is innovative conceptually as this work moves beyond incomplete analyses of only methylation in the CG context in males at the tissue level to a more comprehensive understanding of both mC and hmC in a cell-type specific manner and in both males and females, heretofore completely unexamined factors. Insight into the neuroepigenome with aging is impactful as the epigenome is ?upstream? of all gene and protein expression and may be the best intervention point for pleiotropically beneficial treatments that maintain brain health and function with aging.